Non-toxigenic strain of Aspergillus oryzae and Aspergillus sojae for biocontrol of toxigenic fungi

ABSTRACT

Non-toxigenic strains of Aspergillus such as from the species Aspirgillus oryzae and Aspergillus sojae are useful fungal biocontrol agents for preventing toxin contamination in agricultural commodities, especially those for human consumption such as peanuts and corn. These strains do not produce aflatoxin, any bis-furan ring-containing intermediates of the aflatoxin biosynthetic pathway and cyclopiazonic acid. They are also useful for controlling toxin damage to crops such as cotton. The strains include Aspergillus strains NRRL 21368, NRRL 21369, NRRL 21882, NRRL 30038, NRRL 30039 and mixtures thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel non-toxigenic strains ofAspergillus including Aspergillus flavus (A. flavus), Aspergillusparasiticus (A. parasiticus), Aspergillus oryzae (A. oryzae), andAspergillus sojae (A. sojae); agricultural compositions containingnon-toxigenic strains of Aspergillus flavus (A. flavus), Aspergillusparasiticus (A. parasiticus), Aspergillus oryzae (A. oryzae), andAspergillus sojae (A. sojae) on agriculturally acceptable carriers; andto methods for the control of toxin contamination in agriculturalcommodities using non-toxigenic strains of A. flavus, A. parasiticus, A.oryzae, and A. sojae.

2. Description of the Related Art

Aflatoxins are potent hepatotoxic, carcinogenic compounds produced by A.flavus Link:Fr. and A. parasiticus Speare (CAST, In: Mycotoxins:Economic and Health Risks. Report 116, 99 pp., Council for AgriculturalScience and Technology, 137 Lynn Avenue, Ames, IA 50010). Cyclopiazonicacid (CPA) is another potent mycotoxin that is produced by A. flavus,but not by A. parasiticus. When these fungi invade and grow incommodities such as peanuts, corn, cottonseed, and tree nuts, theresulting contamination with the aflatoxins and CPA often makes thecommodity unfit for consumption. The United States peanut industry hasidentified aflatoxin contamination of peanuts as the number one problemfor which a solution is needed (Consensus Report of the National PeanutCouncil Quality Task Force, 1987, National Peanut Council, Alexandria,Va. 22314). Because peanuts are used primarily for food, strictregulatory limits for the amount of aflatoxin allowable in finishedpeanut products have been established. Although the United States Foodand Drug Administration has an action level of 20 ppb of totalaflatoxins in food products, international tolerances for aflatoxin aremuch lower, typically in the range of 0-4 ppb, and are important becauseU.S. companies compete internationally in the market to export peanutsand peanut products. For this reason the United States peanut industryhas a goal to ensure the delivery of aflatoxin-free peanut products bythe year 2000. Although aflatoxin contamination of peanuts can occurduring postharvest curing and storage, the most significantcontamination usually occurs prior to harvest during periods oflate-season drought stress as peanuts are maturing. The only knownmethod for controlling preharvest aflatoxin contamination in peanuts isirrigation, an option that is unavailable to the majority of peanutgrowers.

Cyclopiazonic acid is an indole-tetramic acid that was first isolatedfrom cultures of Penicillium cyclopium Westling in 1968 (Holzapfel,Tetrahedrom, Volume 24, 2101-2119, 1968). CPA is now know to be producedby a variety of fungi including P. patulum, P. viridicatum, P.puberulum, P. crustosum, P. camemberti, A. flavus, A. versicolor and A.oryzae. In addition, CPA has been found as a natural contaminant of cornand peanuts, often occurring together with aflatoxin (Lansden andDavidson, Applied and Environmental Microbiology, Volume 45,766-769,1983; Urano et al., Journal of AOAC International, Volume 75,838-841, 1992). It was also implicated as the causative agent in a humanintoxication involving consumption of contaminated millet (Rao andHusain, Mycopathologia, Volume 89, 177-180, 1985). With the discovery ofCPA production by A. flavus, 54 isolates of A. flavus were investigatedfor production of CPA and aflatoxin (Gallagher et al., Mycopathologia,Volume 66, 31-36, 1978). It was found that 28 of the 54 (52%) producedCPA whereas only 18 (33%) produced aflatoxin. Regulatory limits for CPAhave not been established; however, because of the co-occurrence ofaflatoxin and CPA in commodities, efforts to attain biological controlof aflatoxin also need to attain control of CPA.

It has been previously found that co-cultivation of either A.parasiticus or A. flavus with species of Penicillium reduce levels ofaflatoxin production while co-cultivation of Fusarium species had nosuch effect (Ehrlich et al., Experiential, Volume 41, 691-693, 1985).These tests did not involve the use of a soil environment.Co-cultivation with A. niger completely eliminated the production ofaflatoxin by a culture of A. flavus (Wicklow et al., Phytopathology,Volume 70, 761-764, 1980). This testing was done under laboratorycontrolled conditions in which the food source involved sterilized corn.

Cotty (U.S. Pat. No. 5,171,86 Dec. 15, 1992 and U.S. Pat. No. 5,294,442Mar. 15, 1994) discloses a non-toxigenic strain of A. flavus whichinhibits aflatoxin production by toxigenic strains. The patent teachesthat agricultural commodities inoculated simultaneously with both anon-toxigenic strain and a toxigenic strain produce seed with up to100-fold less aflatoxin than commodities inoculated with a toxigenicstrain alone. The patent only discloses that the patented strain failsto produce aflatoxin. There is no disclosure of its lack of ability toproduce other toxins such as, for example, CPA.

Cole et al.(U.S. Pat. No. 5,292,661 Mar. 8, 1994) and Dorner etal.(Journal of Food Protection, Volume 55, 888-892, 1992) disclose anon-aflatoxigenic strain of A. parasiticus. The references teach the useof this strain as a biocontrol agent which reduces aflatoxincontamination of soil-borne crops.

Tantaoui-Elaraki (Journal of Environmental Pathology, Toxicology andOncology, Volume 11 (2), 97-101, 1992), Lemke et al. (Applied andEnvironmental Microbiology, July, 1989, 1808-1810), Jishen et al. (ActaAcademiae Medicinae, Volume 8, (1),70-71, February 1986), and Lee(Mycopathologia, Volume 107, 127-130, 1989), all disclose non-toxigenicstrains of A. flavus wherein the strains do not produce aflatoxin.

Horn et al. (Mycologia, Volume 88 (4), 574-587, 1996) discloses isolatesof A. flavus that fail to produce the mycotoxins CPA and aflatoxin.

While various strains of non-toxigenic Aspergillus for control oftoxigenic fungi are known in the art, there still remains a need for aneffective biocontrol agent for toxigenic fungi. The present inventiondescribed below includes non-toxigenic strains of Aspergillus,especially non-toxigenic strains of A. flavus, A. parasiticus, A.oryzae, and A. sojae, which are antagonistic to toxigenic fungi. Thepresent invention also provides a method for controlling toxigenic fungiin agricultural crops which is different from the related art biocontrolagents.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to providebiocontrol agents for the control of toxigenic fungi in plants.

Another object of the present invention is to provide non-toxigenicfungi which are biocontrol agents for preventing contamination of cropswith toxin-producing fungi.

It is another object of the present invention to provide non-toxigenicstrains of fungi selected from A. flavus, A. parasiticus, A. oryzae, andA. sojae as biocontrol agents for preventing contamination of crops withtoxin-producing fungi.

A further object of the present invention is to provide an agriculturalbiocontrol composition containing at least one non-toxigenic strain ofAspergill us, especially A. flavus, A. parasiticus, A. oryzae, and A.sojae.

A still further object of the present invention is to provide a methodfor biocontrol of toxin-producing fungi in plants.

Another object of the present invention is to provide a method forbiocontrol of toxin-producing fungi in plants which includes applyingnon-toxigenic strains of fungi to the soil.

A further object of the present invention is to provide a biocontrolmethod for peanuts, corn and cotton, which includes applyingnon-toxigenic strains selected from A. flavus, A. parasiticus, A.oryzae, and A. sojae and mixtures thereof that do not produce aflatoxinor CPA, to the soil.

Further objects and advantages of the present invention will becomeapparent from the following description.

DEPOSIT OF THE MICROORGANISMS

Aspergillus flavus, designated NRRL 21882 and NRRL 21368; A.parasiticus, designated NRRL 21369; have been deposited under theprovisions of the Budapest Treaty on Nov. 12, 1997 (NRRL 21882), Dec. 8,1994 (NRRL 21368 and NRRL 21369) with the U.S.D.A. Agricultural ResearchService Patent Culture Collection (National Center for AgriculturalUtilization Research, 1815 N. University Street, Peoria, Ill., 61604).Aspergillus oryzae strain S-03, designated NRRL 30038, and Aspergillussojae strain S-12 designated NRRL30039, have been deposited on Jul. 2,1998 under the provisions of the Budapest Treaty with the U.S.D.A.Agricultural Research Service Patent Culture Collection (National Centerfor Agricultural Utilization Research, 1815 N. University Street,Peoria, Ill. 61604).

DETAILED DESCRIPTION OF THE INVENTION

The addition of highly competitive, non-toxigenic strains of A. flavus,A. parasiticus, A. oryzae, and A. sojae as well as mixtures of any ofthe strains, to soil results in lower concentrations of toxins inagricultural commodities. The non-toxigenic strains of Aspergillusbecome biocompetitive with the soil microflora and prevent buildup oftoxin-producing strains that normally occurs during late-season drought.Through biocompetition, the toxigenic strains of fungi found naturallyin soil are replaced by non-toxigenic strains added to the soil.Therefore, any crop subjected to late-season drought stress are invadedpredominately by the biocompetitive strains which are unable to producetoxins.

The method of the invention is applicable to any agricultural commoditywhich is grown for human consumption and/or which is damaged by fungaltoxins such as for example, peanuts, corn, cotton, etc.

For purposes of this invention, a fungal preparation or fungalagricultural biocontrol composition refers to a microbial preparationwherein the microbes comprise, consist essentially of, or consist ofnon-toxigenic strains of Aspergillus. The fungal preparations maycontain one or more of non-toxigenic strains of Aspergillus.Non-toxigenic strains of Aspergillus include any strain which does notproduce the toxins aflatoxin and cyclopiazonic acid (CPA). Theagricultural biocontrol composition for purposes of this inventionincludes a non-toxigenic strain or strains of fungi on agriculturallyacceptable carriers which may be any carrier which the fungi can beattached and are not harmful to the fungi or crops which are treatedwith the composition. Examples of non-toxigenic strains includeAspergillus flavus, A. parasiticus, A. oryzae, and A. sojae. The fungiespecially useful in the present invention are strains possessing theidentifying characteristics of non-toxigenic Aspergillus flavus,designated NRRL 21882 and NIRL 21368; non-toxigenic A. parasiticus,designated NRRL 21369; non-toxigenic A. oryzae, designated NRRL 447,NRRL 552, NRRL 451, NRRL 1730, and S-03; NRRL 30038 and non-toxigenic A.sojae, designated NRRL 3351, NRRL 1988, NRRL 5595, NRRL 5596, NRRL 6271,and S-12 NRRL 30039. These characteristics are the inability to producethe toxins aflatoxin and CPA and the ability to be biocompetitive whenapplied to soils growing agricultural commodities. A. oryzae and A.sojae are "koji molds" that are used in fermentation processes thatresult in the production of soy sauce and other products used for humanconsumption. Strains S-03 and S-12 (NRRL 30038 and 30039, respectively)were obtained commercially from a food manufacturer (Higuchi NatunosukeShoten Co. LTD., 14-2 Harima-cho, Abeno-ku, Osaka, Japan 545).

Non-toxigenic strains of Aspergillus are cultured as single strains ongranular food sources, such as for example rice, rye, etc.; orincorporated into extruded food matrices, such as for example wheatgluten-kaolin matrices. These food sources contain approximately 10⁶colony forming units (CFU) of fungi per gram of food source. Forgranular food sources such as rice or rye, innoculated grains areincubated at about 30° C. for about 4 days, rotating at approximately 2rev/minute while tilted at 70° from horizontal to gently agitate thegrain and prevent fungal sporulation. The inoculated product is storedat about 5° C. until use. For food matrices, conidia are incorporatedinto the matrix, extruded, cut into lengths that can be easily appliedwith standard farm equipment and dried for storage at about 5° C.

The non-toxigenic strains of Aspergillus are applied to soil in amountseffective to reduce toxin levels in agricultural commodities. As usedherein "reduce toxin levels" refers to a reduction in amounts of toxincompared to that which would be expected in agricultural commoditieswhich were not treated according to the methods of the presentinvention. Any accurate method of measuring and comparing toxin levelsmay be used for such comparisons, as would be apparent to those skilledin the art. As used herein "in amounts effective", "an amount effective"or "an effective amount" refer to the amount of the fungal preparationadministered wherein the effect of the administration acts to reducetoxin contamination of agricultural commodities. The granular orextruded products are applied to the soil at a rate of approximately 20to 900 pounds per acre, with 100-200 pounds per acre preferred, and20-50 pounds per acre most preferred, when the foliage canopy is about12 inches wide. The soil surface under the canopy provides a humid,protected environment which promotes growth and sporulation of thenon-toxigenic fungi. The strains can be applied as single straincompositions or the dried products can be mixed in about equalproportions to provide a composition made up of different strains ofAspergillus.

The following examples are intended to further illustrate the inventionand are not intended to limit the scope of the invention as defined bythe claims.

EXAMPLE 1

Strains of A. flavus were isolated from peanuts grown in theEnvironmental Control Plot Facility at the National Peanut ResearchLaboratory during crop year 1991. Randomly selected peanuts, which weregrown under drought stress conditions conducive to invasion of peanutswith Aspergillus and contamination with aflatoxin, were surfacesterilized with a 2.0% solution of sodium hypochlorite and incubated onCzapek agar plates at 25° C. for 7 days. Fungal colonies that grew fromthe peanuts were transferred to Czapek agar slants and identified as togenus and species. Numerous isolations of A. flavus and A. parasiticuswere made. Strains were maintained on Czapek agar slants and as dryconidia on silica gel. In a screening program (see example 4 for detailsof the screening program) to identify non-toxigenic strains of A. flavusfor possible use as biocontrol agents against aflatoxin contamination ofpeanuts, it was found that a particular strain of A. flavus (NPL 45;NRRL 21882) was not only incapable of producing aflatoxin, but alsoincapable of producing CPA, another mycotoxin commonly produced by A.flavus.

EXAMPLE 2 SCREENING FUNGAL ISOLATES FOR NON-TOXIGENIC STRAINS

A screening program was developed to identify non-toxigenic strains ofA. flavus and A. parasiticus for possible use as biocontrol agentsagainst aflatoxin contamination of peanuts. All fungi included in thescreening program were cultured in vials for 4 days at 30° C. on aliquid medium containing glucose, soytone, yeast extract, and sucrose(Horn et al., Mycologia, Volume 88 (4), 574-587, 1996; hereinincorporated by reference). Vial cultures were extracted by adding 1 mLof chloroform to each vial, breaking up the fungal material with aspatula, and vortexing for 30 sec. Vials were allowed to sit undisturbedfor several minutes to allow the chloroform and aqueous layers toseparate. Occasionally, centrifugation was required to achieveseparation of the two layers. For aflatoxin analysis, an aliquot of thechloroform layer was transferred to a 4 mL HPLC autosampler vial,evaporated to dryness under a stream of nitrogen, and redissolved inHPLC injection solvent consisting of methanol:water:acetic acid(62:38:0.1, v/v/v). Aflatoxins were quantified by reversed-phase HPLCanalysis with a Waters Nova-Pak C₁₈ column, a mobile phase ofwater:methanol:butanol (70:35:0.6,v/v/v), and fluorescence detection.Fluorescence of aflatoxins B₁ and G₁ was enhanced by postcolumnphotochemical derivatization with a photochemical reactor (Joshua,Journal of Chromatography A, Volume 654, 247-254, 1993). For analysis ofCPA and other aflatoxin biosynthetic pathway intermediates, anotheraliquot of the chloroform layer was transferred to a 4 mL vial,evaporated to dryness under a stream of nitrogen, and redissolved inHPLC mobile phase consisting of n-heptane:2-propanol:water:40%tetrabutylammonium hydroxide (2560:1120:32:8,v/v/v/v/). CPA and othermetabolites were separated on a Zorbax Rx-Sil column and detected byphotodiode array. Metabolite standards that cultures were screenedagainst included CPA, O-methyl-sterigmatocystin, sterigmatocystin,norsolorinic acid, averufin, averantin, and kojic acid.

Aspergillus flavus strains NRRL 21882 (NPL 45), NaRL 21368 (AFCM), NRRL18543 (AF36), P61, P84, and F48; A. parasiticus strain NRRL 21369 (CM2);A. oryzae NRRL 447, NRRL 552, NRRL 451, NRRL 1730, and NRRL 30038 (S-03)and A. sojae NRRL 3351, NRRL 1988, NRRL 5595, NRRL 5596, NRRL 6271, andNRRL (S-12) were cultured as described above and analyzed for productionof aflatoxin, CPA, and other metabolites listed above. Strains P61, P84and F48 are known aflatoxin and/or CPA producers and are routinelyincluded in the screening process as positive controls.

Results of the analyses for some of the cultures for aflatoxin and CPAare shown in Table 1 below. Strain AF36 (NRRL 18543), as well as thethree strains known to produce CPA, produced CPA. All strains producedkojic acid. No intermediates (containing the bis-furan ring system) inthe aflatoxin biosynthetic pathway were detected in any of the isolates.Only trace amounts (<0.1 μg/ml) of aflatoxin production occurred withany of the NRRL isolates.

                  TABLE 1                                                         ______________________________________                                        Biocompetitive Agent Metabolite Screen.                                       CPA Production by Isolates of A. flavus                                       Strain         Aflatoxin (μg/ml)                                                                      CPA (μg/ml)                                     ______________________________________                                        AFCM-NRRL 21368                                                                              0.002       0                                                  NPL45-NRRL 21882                                                                             0.019       0                                                  P 61           0.008       42.2                                               P 84           0.066       46.1                                               F 48           227.950     47.2                                               AF 36-NRRL 18543                                                                             0.001       38.7                                               ______________________________________                                    

EXAMPLE 3

A. flavus, strain NRRL 21882 (NPL 45), was irradiated under ultravioletlight to produce color mutant strains that lack the capability ofproducing aflatoxins, CPA, or known toxic intermediates in the aflatoxinbiosynthetic pathway. A color mutant was isolated (NRRL 21368) that wasincapable of producing aflatoxins, CPA, or known toxic intermediates inthe aflatoxin biosynthetic pathway.

A color mutant strain of A. parasiticus (NRRL 6111) that is a knownproducer of aflatoxins and an early biosynthetic precursor to aflatoxin,norsolorinic acid, was irradiated under ultraviolet light to produceother mutant strains that lack the capability of producing aflatoxins,CPA, or known toxic intermediates in the aflatoxin biosynthetic pathway.A color mutant was isolated (NRRL 21369) and found to be incapable ofproducing aflatoxins, norsolorinic acid, other known intermediates inthe aflatoxin biosynthetic pathway, or CPA.

EXAMPLE 4 FIELD DELIVERY OF BIOCOMPETITIVE STRAINS

Rice inoculum was prepared by culturing each strain on autoclaved,long-grain rice in 2800-ml Fernbach flasks (500 g of rice with 150 mldistilled water). Rice was inoculated with 1 ml of a conidial suspension(106/ml) and incubated at 30° C. for 4 d on a rotating platform (2rev/min) tilted 70° from horizontal to gently agitate the rice andprevent fungal sporulation. Rice inoculum was then dried in a shallowpan in a forced-air draft oven at 50° C. for 6 h or until the moisturecontent was ≦7%. Rice inoculum was stored at 5° C. until used.

Alternatively, inoculum in the form of pesta was prepared by the methodof Connick et al. (Biological Control, Volume 1, 281-287,1991). Conidiaof NRRL 21368 (A. flavus) and NRRL 21369 (A. parasiticus) wereincorporated into a wheat gluten-kaolin matrix by extrusion. Extrudedproduct (pesta) was cut into 1-2 mm lengths and dried for storage priorto use.

For field application, inoculum was placed in a Gandy box fitted to atractor and banded over the peanut row, optimally at 40-50 days afterplanting, or when the width of a peanut row (measured from the outeredges of the foliage canopy) is about 12 inches. The inoculum filtersthrough the canopy of foliage and comes to rest in a humid, protectedenvironment on the soil surface under the canopy. Uptake of moisture bythe inoculum granules results in growth of the incorporatedbiocompetitive fungi, which produces an abundance of conidia on thesurface of the granules. As the conidia are dispersed over the soilsurface, full delivery of the biocompetitive fungi is realized.

EXAMPLE 5

Biocompetitive agents, NRRL 21882 (A. flavus, NPL 45), NRRL 18991 (A.parasiticus; see U.S. Pat. No. 5,292,661--Mar. 8, 1994), NRRL 21368 (A.flavus color mutant of NRRL 21882), and NRRL 21369 (A. parasiticus colormutant), were tested in the NPRL environmental control plot facility forcontrol of preharvest aflatoxin contamination of peanuts during cropyear 1992. Plots consisted of six rows of cv. Florunner peanuts (6 mlong spaced 0.9 m apart). Plots were inoculated with the biocompetitiveagents by distributing fungus-colonized rice inoculum on soil aroundpeanut plants at approximately 45 days after planting at a rate of 200pounds per acre. Treatments consisted of NRRL 21882 alone, NRRL 18991alone, NRRL 21882 and NRRL 18991 in combination, and a combination ofNRRL 21368 and NRRL 21369. Equivalent plots were also left untreated toserve as controls. Harvested peanuts were shelled and kernels were sizedinto commercial categories including jumbo, medium, number 1, soundsplits, and oil stock. Visibly moldy and damaged kernels werehand-picked from the jumbo, medium, number 1, and sound splitscategories (edible categories) for analysis as a separate category. Allpeanuts were subjected to aflatoxin analysis with no subsampling usingthe high-performance liquid chromatography method of Dorner and Cole (J.Assoc. Off. Anal. Chem., Volume 71, 43-47, 1988; herein incorporated byreference).

Results of aflatoxin analyses of peanuts at harvest are shown below inTable 2. Aflatoxin concentrations for the combined edible (jumbo,medium, number 1, and sound splits), inedible (oil stock and damagedkernels), and total kernels categories were calculated by dividing thetotal weight of toxin (ng) for the combined categories by the totalpeanut weight (g) for those combined categories. Reductions in aflatoxincontamination as a result of treatment with the biocompetitive agentsranged from 39.2% in inedible category peanuts from the treatment withthe combination of A. flavus and A. parasiticus color mutants (NRRL21368+NRRL 21369) to 98.9% in edible category peanuts from the treatmentwith the combination of wild-type isolates of A. flavus and A.parasiticus (NRRL 21882+NRRL 18991). A higher degree of control wasusually seen in the more important edible category peanuts with theexception of the treatment with A. parasiticus NRRL 18991 alone.

                  TABLE 2                                                         ______________________________________                                        Aflatoxin contamination (ppb) of peanuts treated with                         various combinations of biocompetitive agents during crop year 1992.                      Peanut Category                                                   Treatment     Edible     Inedible Total                                       ______________________________________                                        Control       171.1      2188.2   354.9                                       NRRL 21882    9.7        685.1    71.2                                        % Reduction   94.3%      68.7%    79.9%                                       Control       278.7      5901.4   579.5                                       NRRL 18991    76.4       434.1    108.1                                       % Reduction   72.6%      92.6%    81.3%                                       Control       75.1       6465.9   525.2                                       NRRL 21882 + 18991                                                                          0.8        273.7    44.3                                        % Reduction   98.9%      95.8%    91.5%                                       Control       147.4      2072.3   407.4                                       NRRL 21368 + 21369                                                                          26.8       1260.8   151.0                                       % Reduction   81.8%      39.2%    62.9%                                       ______________________________________                                    

EXAMPLE 6

In crop year 1993, a study similar to that described in Example 5 wascarried out except that treatment with the individual strains, NRRL21882 and NRRL 18991, was not done. Treatments included the combinationof NRRL 21882 plus NRRL 18991 and NRRL 21368 plus NRRL 21369. Results,shown below in Table 3, again showed better control in the ediblecategory peanuts and better overall control by the wild-type isolates(NRRL 21882 plus NRRL 18991).

                  TABLE 3                                                         ______________________________________                                        Aflatoxin contamination (ppb) of peanuts treated with                         various combinations of biocompetitive agents during crop year 1993.                      Peanut Category                                                   Treatment     Edible     Inedible Total                                       ______________________________________                                        Control       89.5       3102.7   361.8                                       NRRL 21882 + 18991                                                                          3.2        449.1    49.2                                        % Reduction   96.5%      85.5%    86.4%                                       Control       100.1      1192.0   242.9                                       NRRL 21368 + 21369                                                                          5.3        603.6    84.1                                        % Reduction   94.7%      49.4%    65.4%                                       ______________________________________                                    

EXAMPLE 7

Biocompetitive agents, NRRL 21882 (A. flavus, NPL 45) and NRRL 18543 (A.flavus, AF36), were field-tested for control of preharvest aflatoxin andCPA contamination of peanuts. Plots (15 m×6 rows, spaced 0.9 m apart) ofcv. Florunner peanuts were grown in an agricultural field in TerrellCounty, Georgia, during crop years 1996 and 1997. Plots were inoculatedwith the biocompetitive agents by distributing fungus-colonized riceinoculum on soil around peanut plants at approximately 45 days afterplanting at a rate of 200 pounds per acre in 1996 and 20 pounds per acrein 1997. Four plots were separately treated with each biocompetitivestrain alone and four untreated plots served as controls. Plots thatwere treated with a particular biocompetitive fungus in 1996 weretreated with the same biocompetitive fungus in 1997.

Peanuts were harvested with conventional peanut-harvesting equipment.Peanuts were shelled and kernels were sized into commercial categoriesincluding jumbo, medium, number 1, sound splits, and oil stock. Visiblymoldy and damaged kernels were hand-picked from the jumbo, medium,number 1, and sound splits categories (edible categories) for analysisas a separate category. Each size category (including damaged) wasanalyzed for aflatoxin and CPA. Peanuts were prepared for analysis bygrinding to a paste in a vertical cutter mixer. Aflatoxin analyses werecarried out on 200 g of the ground paste using the HPLC method cited inExample 5. CPA analyses were carried out on 50 g of ground paste byextraction with methanol:1% sodium bicarbonate (70:30, v/v). The extractwas filtered and purified on a CPA-immunoaffinity column. Eluate fromthe column was subjected to HPLC analysis using the system described inExample 2 above for fungal cultures. After aflatoxin and CPAconcentrations were determined for each category, concentrations foredible peanuts (jumbo, medium, and number 1 categories) and all peanuts(all categories) were calculated by dividing the total weight of toxin(ng) for the combined categories by the total peanut weight (g) forthose combined categories.

Results for edible category peanuts and all peanuts combined are shownbelow in Table 4. The treatment of peanut soil with NPL 45 (NRRL 21882)resulted in a mean reduction in aflatoxin of edible peanuts of 79.5%,whereas treatment with AF36 (NRRL 18543) resulted in a mean reduction of61.4%. The reduction in all peanuts averaged 27.2% for treatment withNPL 45 and 26.3% for treatment with AF36. CPA contamination of peanutsin control plots was relatively low under these conditions, buttreatment with NPL 45 resulted in no contamination of edible peanutswith CPA and a 39% reduction in CPA levels in all peanuts. In contrast,treatment with the CPA-producing fungus, AF36, resulted in largeincreases in CPA contamination in both edible peanuts as well as allpeanuts.

                  TABLE 4                                                         ______________________________________                                        Aflatoxin and Cyclopiazonic Acid in Peanuts.                                             Edible Peanuts                                                                            All Peanuts                                            Treatment    Aflatoxin                                                                              CPA      Aflatoxin                                                                            CPA                                     ______________________________________                                        Control                                                                              1         8.4      0.0    195.3  18.2                                         2         44.0     3.7    44.8   3.1                                          3         5.0      3.1    18.9   6.4                                          4         27.7     0.0    156.4  8.9                                          Mean      21.5     1.7    103.9  8.7                                   NPL45  1         1.4      0.0    155.4  9.7                                          2         7.5      0.0    124.1  7.0                                          3         0.0      0.0    2.2    0.0                                          4         8.6      0.0    20.8   4.3                                          Mean      4.4      0.0    75.6   5.3                                   AF 36  1         0.5      191.4  35.3   760.2                                        2         11.5     18.5   160.3  100.2                                        3         21.1     0.0    90.5   82.6                                         4         0.1      69.2   20.2   344.3                                        Mean      8.3      69.8   76.6   321.9                                 ______________________________________                                    

EXAMPLE 8

Biocompetitive agents NRRL 21882 (A. flavus), NRRL 21368 (A. flavus),and NRRL 21369 (A. parasiticus) were field-tested for control ofaflatoxin and CPA contamination over the course of a three-year period(1995-1997). In 1995 soil was inoculated with 200 pounds per acre of ryeseed that had been colonized with NRRL 21368 and NRRL 21369. In 1996soil inoculum in the form of pesta included a combination of NRRL 21368(A. flavus), and NRRL 21369 (A. parasiticus) applied at a rate of 200pounds per acre to 1.5 acres of peanuts. An equivalent area of peanutswas not treated and served as controls. In 1997 the testing wascontinued by treating the same soil with a mixture of NRRL 21882 (A.flavus) and NRRL 21369 (A. parasiticus). In 1995 and 1996, environmentalconditions were not conducive for preharvest aflatoxin contamination,and peanuts (both from treated and control plots) were not contaminated.However, in 1997 a significant drought occurred near the end of thegrowing season, thus providing conditions that were conducive forcontamination to occur. Peanuts were harvested with conventionalequipment and placed in conventional peanut drying wagons for transportto a Federal-State Inspection Service sampling station. A pneumaticprobe was used to draw 15 samples from each wagon containing peanutsfrom the treated and untreated areas. The samples were composited by theautomatic sampler, and they were taken to the laboratory where they wereriffle-divided into eight separate samples. Each sample was shelled andsized into commercial size categories as described in Example 5 withdamaged kernels being removed from the edible categories. All peanutsfrom each category were extracted and analyzed for aflatoxin.Subsequently, another eight samples were taken from each group oftreated and untreated peanuts and analyzed for CPA. Concentrations ofaflatoxin and CPA for all peanuts were calculated as in Example 5 bydividing the total weight of toxin (ng) for the combined categories bythe total peanut weight (g) for those combined categories.

Results are presented below in Table 5. The average reduction inaflatoxin in treated peanuts compared to controls was 91.6%, and theaverage reduction in CPA in treated peanuts compared to controls was85.7%.

                  TABLE 5                                                         ______________________________________                                        Aflatoxin and CPA contamination (ppb) of field-grown                          peanuts in 1997 and treated with a combination of biocompetitive              agents including NRRL 21882 (A. flavus) and NRRL 21369 (A.                    parasiticus color mutant).                                                                  Aflatoxin                                                                            CPA                                                      ______________________________________                                        Control         603.5    30.7                                                 Treated         50.8     4.4                                                  % Reduction     91.6%    85.7%                                                ______________________________________                                    

EXAMPLE 9 AFLATOXIN REDUCTION IN CORN

Soil in eight corn plots (5.5×24.4 m) was inoculated with formulationsincluding NRRL 21368 (A. flavus), and NRRL 21369 (A. parasiticus) duringcrop years 1994-1996. In 1997 the same plots were inoculated with NRRL21882 (A. flavus) and NRRL 21369 (A. parasiticus). Eight corn plots in aseparate part of the field were not inoculated and served as controls.Corn from each plot was harvested with a conventional combine andbagged. Corn was ground with a Romer subsampling mill and analyzed foraflatoxin by HPLC as cited in Example 5.

In 1994 and 1995, aflatoxin contamination did not occur to anysignificant degree, probably because environmental conditions in thoseyears were relatively cool and wet. In 1996, the aflatoxin concentrationin corn from treated plots averaged 23.6 ppb, a significant reduction(P<0.001) compared with the aflatoxin in control plots, which averaged188.3 ppb. This coincided with a significantly (P=0.018) reducedcolonization of corn by wild-type A. flavus in treated plots (1.8% ofkernels) compared with control plots (5.4%). In 1997, aflatoxin wasagain significantly reduced (P=0.024) in treated corn (29.8 ppb)compared with untreated corn (87.5 ppb). Treated corn was predominatelycolonized by the introduced strain of A. flavus (NRRL 21882)(26.7% ofkernels) compared with wild-type A. flavus (2.9%).

EXAMPLE 10 AFLATOXIN REDUCTION BY ASPERGILLUS ORYZAE AND ASPERGILLUSSOJAE

Aspergillus oryzae and A. sojae are species that are closely related toA. flavus and A. parasiticus. Several strains of these fungi werecultured on rice as in Example 5 and tested in environmental controlplots (Example 5) for biological control of aflatoxin contamination.Strains of A. oryzae tested included NRRL 447, NRRL 552, NRRL 451 andNRRL 1730. Strains of A. sojae, tested were NRRL 3351, NRRL 1988, NRRL5595, NRRL 5596 and NRRL 6271. Each strain was cultured separately andthe rice cultures were mixed in approximately equal amounts based onweight before application to the soil. Strain NRRL 451 was mixed in athalf the weight of the other strains. The mixture was applied at a rateof 960 lbs./acre on two plots (See Example 5). Two untreated plotsserved as controls.

Results of aflatoxin analyses of peanuts at harvest are shown below inTable 6. Mixtures of A. oryzae and A. sojae reduced the concentration ofaflatoxin, particularly in the edible category peanuts.

                  TABLE 6                                                         ______________________________________                                                  Peanut Category                                                     Treatment   Edible       Inedible                                                                              Total                                        ______________________________________                                        Control     640.3        9709.5  1472.4                                       A. oryzae/A. sojae                                                                        9.7          3167.6  234.0                                        % Reduction 98.5         67.4    84.1                                         ______________________________________                                    

The foregoing detailed description is for the purpose of illustration.Such detail is solely for that purpose and those skilled in the art canmake variations therein without departing from the spirit and scope ofthe invention.

We claim:
 1. A non-toxigenic fungal biocontrol agent comprising abiologically pure Aspergillus strain selected from the group consistingof Aspergillus oryzes, Aspergillus sojae and mixtures thereof; whereinsaid strain does not produce aflatoxin, any bis-furan ring-containingintermediates in the aflatoxin biosynthetic pathway, and cyclopiazonicacid.
 2. An agricultural biocontrol composition comprising abiologically pure non-toxigenic strain of Aspergillus selected from thegroup consisting of Aspergillus oryzae, Aspergillus sojae and mixturesthereof; wherein said strain does not produce aflatoxin, any bis-furanring-containing intermediates in the aflatoxin biosynthetic pathway, andcyclopiazonic acid, and an agriculturally acceptable carrier.
 3. Anon-toxigenic fungal biocontrol agent consisting essentially of abiologically pure Aspergillus strain having all of the identifyingcharacteristics of a strain selected from the group consisting of NRRL21368, NRRL 2136g, NRRL 21882, NRRL 30038, NRRL 30039, and mixturesthereof.
 4. An agricultural biocontrol composition consistingessentially of a biologically pure Aspergillus strain having all of theidentifying characteristics of a strain selected from the groupconsisting of NRRL 21368, NRRL 21369, NRRL 21882, NRRL 30038, NRRL30039, and mixtures thereof; and an agriculturally acceptable carrier.5. A method for reducing toxin contamination of agricultural commoditiescomprising applying an agricultural biocontrol composition containing abiologically pure strain of Aspergillus having all of the identifyingcharacteristics of an Aspergillus strain selected from the groupconsisting of NRRL 21368, NRRL 21369, NRRL 21882, NRRL 30038, NRRL30039, and mixtures thereof with an agriculturally acceptable carrier;to soil around plants, wherein said Aspergillus strain does not produceaflatoxin, any bis-furan ring-containing intermediates of the aflatoxinbiosynthetic pathway, and cyclopiazonic acid.
 6. The method of claim 5wherein said agricultural commodities are selected from the groupconsisting of peanuts, corn, cotton, and tree nuts.
 7. A method forreducing toxin contamination of agricultural commodities comprisingapplying an agricultural biocontrol composition containing abiologically pure Aspergillus strain selected from the group consistingof Aspergillus oryzae, Aspergillus sojae, and mixtures thereof; with anagriculturally acceptable carrier; to soil around plants, wherein saidAspergillus strain does not produce aflatoxin, any bis-furanring-containing intermediates of the aflatoxin biosynthetic pathway, andcyclopiazonic acid.
 8. The method of claim 7 wherein said agriculturalcommodities are selected from the group consisting of peanuts, corn,cotton, and tree nuts.